J Food Sci Technol (July 2017) 54(8):2203–2213
DOI 10.1007/s13197-017-2614-8
ORIGINAL ARTICLE
Effects of microbial transglutaminase on physicochemical
properties, electrophoretic patterns and sensory attributes
of veggie burger
Zahra Forghani1 • Mohammad Hadi Eskandari1 • Mahmoud Aminlari2
Seyed Shahram Shekarforoush3
Revised: 7 January 2017 / Accepted: 28 March 2017 / Published online: 19 June 2017
Ó Association of Food Scientists & Technologists (India) 2017
Abstract The main objective of this study was to inves-
tigate the effects of microbial-transglutaminase (MTGase
0–0.75%)/sodium-caseinate (SC 0–2%) as crosslinker
agents on proximate analysis, binding properties (express-
ible moisture and shrinkage), texture analysis, elec-
trophoretic patterns, instrumental color, and sensory
properties of veggie burgers. Addition of SC and MTGase
positively affected shrinkage and expressible moisture. It
also increased hardness, springiness, chewiness, and cut-
ting-force of burgers. Presence of SC had no effects on
cohesiveness of burgers. Total protein and ash of samples
were increased by treatment with SC. The lightness (L*) of
samples was significantly decreased by 0.75% MTGase.
No significant influence of SC on samples color parameters
was observed. The results indicated that distinct protein
bands were not formed on the SDS–PAGE of burger
Electronic supplementary material The online version of this
article (doi:10.1007/s13197-017-2614-8) contains supplementary
material, which is available to authorized users.
& Mohammad Hadi Eskandari
Eskandar@shirazu.ac.ir
Zahra Forghani
zahra.forghani@yahoo.com
Mahmoud Aminlari
aminlari@shirazu.ac.ir
Seyed Shahram Shekarforoush
shekar@shirazu.ac.ir
1
Department of Food Science and Technology, School of
Agriculture, Shiraz University, Shiraz, Iran
2
Department of Biochemistry, School of Veterinary Medicine,
Shiraz University, Shiraz, Iran
3
Department of Food Hygiene, School of Veterinary
Medicine, Shiraz University, Shiraz, Iran
•
samples and resulted in a smearing pattern on the gel.
When soy-protein was incubated with MTGase, a pro-
gressive decrease in the intensity of the bands corre-
sponding to the subunits 7S and 11S globulins was
observed concomitant with disappearance of A3 and B3
bands. Electrophoresis pattern of gluten was slightly
changed after MTGase treatment. There were significant
differences in color, taste, appearance, mouth feel, and
overall acceptability between treated and control samples.
Results suggest that production of veggie burgers using
MTGase alone or in combination with SC brings about
covalent cross-linking between homologous and heterolo-
gous proteins to form high-molecular weight polymers,
thereby improving the mechanical properties of veggie
burgers and profoundly increases the acceptability of the
end product.
Keywords Electrophoresis  Microbial-transglutaminase 
Sodium-caseinate  Texture  Veggie burger
Introduction
The Institute of Medicine suggests that human adults need
a minimum of 0.8 g protein for every kilogram of body
weight per day (Johnson 2013). Red meat is a major food
source of protein, but many studies have shown that higher
consumption of red meat is linked to increased risk of
cancers, diabetes, and cardiovascular diseases. The use of
healthier protein sources, especially plant sources in place
of red meat, can lower the risk of these diseases. In addi-
tion, the rise in the number of vegetarians necessitates the
increased use of plant sources of protein, such as grains,
nuts, and seeds (Pan et al. 2012).
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The change in eating pattern requires new products which
are attractive to consumers but does not deprive them of the
nutrients normally supplied by meat products (Wild et al.
2014). Soy protein and wheat gluten are the most common
raw materials for meat-like products (Ahirwar et al. 2015).
The nutritional benefits and functional properties of plant
proteins make them good candidates for the manufacture of
healthy fast foods. The development of these new products as
a substitute for meat that are acceptable to the consumers is a
challenge for food producers (Van Trijp and Van Kleef
2008). The textural characteristics of meat analogues, such
as softness and pasty texture, incohesive and poor chewiness
are critical limiting factors in consumer’s acceptance of these
products (Baer and Dilger 2014).
Meat industry is efficiently using different texturizing
agents such as alginate, casein, fibrinogen and thrombin,
transglutaminase, etc. to improve textural attributes, water-
holding capacity and appearance of restructured meat
products (Hong and Xiong 2012). Microbial transglutami-
nase (MTG or MTGase) catalyzes covalent cross-link for-
mation between primary amines (such as e amino group of
lysine) and the amide group of glutamine residues to form
high molecular weight polymers, resulting in improvement
of mechanical properties. MTGase is used as a texturizing
agent in several meat products, such as sausages (Baer and
Dilger 2014), burgers (Velioğlu et al. 2010), and restructured
meats (Kilic 2003; Herranz et al. 2013) to enhance the net-
work structure of the products. Nevertheless, few studies
have dealt with the possible influence of the application of
MTGase/caseinate as a crosslinking agent on the textural
properties of veggie burgers. Previous studies have shown
that casein is the best substrate for MTGase (Kilic 2003) and
has good potential for improving texture of meat analogues,
Therefore, the main objective of this work was to investigate
the effects of MTGase/SC on the improvement of physico-
chemical, electrophoretic patterns and sensory properties of
veggie burgers.
Materials and methods
Materials
Textured soy protein (TSP) (Sonic Co., India.), spices
(Meat Creaks, Germany), carrageenan and xanthan (BLG,
China) and sodium caseinate (Pegah Fars Dairy, Shiraz,
Iran) were purchased. Gluten, hydrogenated soybean oil,
sunflower oil, and other ingredients (onion, turmeric, and
baking powder) were also purchased from the local market
(Shiraz, Iran). MTGase was from Ajinomoto Co. (ACTIVA
WM, Ajinomoto Europe Sales GmbH, Germany). The
composition of commercial enzyme was 1% of MTGase
and 99% maltodextrins (activity of &96 U/g).
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Preparation of veggie burgers
Figure 1 shows the flow chart for preparation of veggie
burgers. When all of the ingredients were weighted, an
initial mixture containing water soaked TSP and hydro-
genated vegetable oil were ground in a grinder with a
5 mm diameter mesh (Pars-Khazar Co., Model MT1200,
Iran) and then were mixed in a mixer (Pars-Khazar Co.
Model GMT-05 B2, Iran) for 2 min. Powdered ingredi-
ents were subsequently added to the mass and mixed for
10 min (Fig. 1). Then the mixture was divided into 12
portions for further implementing 12 types of treatment
plan. MTGase solution was prepared as described in
Ajinomoto Co. manual. MTGase (at four levels: 0, 0.25,
0.5, and 0.75%) and sodium caseinate (at three levels: 0,
1, and 2%) were added and mixed for 3 more minutes.
One hundred gram portions of the pastes were machine-
shaped into burgers. The burgers were stored at room
temperature for 1 h, after which the samples were
packed in PE/PA (Polyethylene/Polyamide) 75 micron
plastic bags and stored at 4 °C for 24 h to complete the
enzyme activity. All analyses were performed in three
replications.
Proximate analysis
The content of protein (AOAC 981.10), moisture (AOAC
950.46), ash (AOAC 992.16), total fat (AOAC 991.36), and
dietary fiber (AOAC 992.16) was determined using AOAC
method (Horwitz and Horwitz 2000).
pH of raw veggie burgers was measured by making a
10% (w/v) suspension of each sample in distilled water. pH
was determined with a portable pH meter (Model 501-05
Barcelona, Spain) in triplicate (Dzudie et al. 2002).
Evaluation of binding properties
Expressible moisture (EM)
EM was determined using a press technique as described
by Wang and Zayas (1992) with slight modification. In this
study digital image processing was used to determine the
expressible moisture of burgers. A 0.3 g sample was placed
between 2 filter papers (Whatman No. 2) and was pressed
between 2 glass plates under 1 kg weight. After 20 min,
the upper plate was removed and photos were taken from
the filter papers (Wang and Zayas 1992). The area of the
pressed batter and the total area of the moistened paper
(cm2) from the digital pictures were measured using
Digimizer, an image analysis software (Version 4.3.5,
MedCalc Software, Belgium). EM was expressed as the
percentage ratio of moisture released from the sample to
the initial sample weight.
J Food Sci Technol (July 2017) 54(8):2203–2213
Fig. 1 Flow chart and variables involved in the experimental design for the veggie burger production
Shrinkage
Shrinkage was measured using an image processing
method (Velioğlu et al. 2010). The image size was
1900 9 1900 pixels and the pictures included two parts,
black (background) and white (veggie burger). Images
were processed using Matlab R2015a (The MathWorks,
Inc., Natick, MA) to provide the areas of burgers before
and after cooking. Percentages of shrinkage were calcu-
lated using the following formula:
Shrinkage ð%Þ ¼ ½ðArea of raw burgers  Area of
cooked burgersÞ=Area of raw burgers 100
Texture analysis
The effect of adding MTGase and SC on the texture of
veggie burgers was studied using a texture analyzer
(Brookfield, CT3, UK). Burgers were equilibrated to room
temperature for 1 h and cut into small cubes
(1 9 191 cm). Samples were compressed to 70% of their
original height in two cycles with 2 s intervals between
cycles. Crosshead speed of 20 cm/min was performed by
cylindrical aluminum probe (diameter: 10 cm). Hardness
2205
[peak force on first compression (g)], cohesiveness (ratio of
the active work done under the second force–displacement
curve to that done under the first compression curve),
springiness [distance the sample recovered after the first
compression (mm)], and chewiness [hardness 9 cohe-
siveness 9 springiness (g mm)] were determined from the
resulting force/deformation curves (Canto et al. 2014).
Nine samples per treatment were measured. The load cell
capacity was set to 250 N.
Instrumental cutting force (g) determinations were made
on a total of 9 veggie cooked burgers per replication.
Burgers were fried for 7 min (3.5 min per side) using 9 g
oil at medium intensity flame. After cooling to 23 °C each
sample was cut into 6 9 2.5 cm sections. Cutting force of
each piece of burger was measured using texture analyzer
with Warner–Bratzler blade (Berry and Bigner 1996; Baer
and Dilger 2014). Cut speed was 200 mm/min. The load
cell capacity used was 1000 N.
Instrumental color evaluation
The cooked samples were equilibrated to laboratory tem-
perature (23 ± 2 °C) and then L* (lightness), a* (redness
‘‘?’’ or greenness ‘‘-’’), and b* (yellowness‘‘?’’ or
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blueness ‘‘-’’) of samples were measured by a computer
vision system according to the method described by
Girolami et al. (2013). A 7.1-megapixel digital camera
(Canon, Ixus70, Japan) was used for photographing and the
camera setting was adjusted to the followings: shutter
speed 1/6 s, manual operation mode and flash off. Lighting
was achieved with four fluorescent lamps (Philips Master
Graphica TLD 965). The camera was located vertically at a
distance of 30 cm from the sample. Lighting was achieved
with four fluorescent lamps (Philips Master Graphica TLD
965). Adobe Photoshop CS3 software was employed for
image analysis. The size of samples was 6 9 2.5 cm
(Girolami et al. 2013).
Protein–protein interactions (electrophoresis
analysis)
Slab sodium dodecyl sulfate–polyacrylamide gel elec-
trophoresis (SDS–PAGE) was performed based on the dis-
continuous buffer system of Laemmli with some
modifications as described by Aminlari and Majzoobi 2002.
Frozen samples (burger batters, soy protein, and gluten)
were allowed to thaw at room temperature and were sub-
jected to SDS–PAGE (Aminlari and Majzoobi 2002). A
12% acrylamide separating gel (width 9 height 9 thick-
ness = 14 mm 9 14 mm 9 1 mm), and a 4% acrylamide
stacking gel was used. Treated protein samples (0.2% pro-
tein concentration) were mixed with an equal volume of
SDS–PAGE sample buffer (4% SDS, 20% glycerol, 10% b-
mercaptoethanol in 0.125 M Tris HCl, pH 6.8) and heated in
boiling water for 5 min. Aliquots of 30 lg of protein per
well were loaded onto the gel. The intensity of color for each
band and their protein content were determined by ImageJ
software (USA, version 1.46r).
Sensory evaluations
Sensory evaluations were carried out by 20 trained pan-
elists from the staff of Setareh Yakhi Asia, a food pro-
cessing and marketing company (Ice-Asian co-star, BA
Food, Shiraz, Iran, site: http://www.bafoods.com/). Ten
women and ten men, aged between 20 and 35 years old,
tested the veggie burgers for appearance, aroma, texture,
overall acceptability, and mouth feel. Panelists were
trained according to the guidelines of American Meat
Science Association. The degree of like or dislike for
samples was evaluated by acceptance test. Samples were
fried and served warm to the panelists. Panelists received
the samples in a randomized order (based on random
3-digit numbers). Unsalted crackers and water were given
between samples to clear the mouth of residual flavor
(Meilgaard et al. 2006).
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Statistical analysis
All data were statistically analyzed using the one-way
analysis of variance (ANOVA) procedure of the SAS 9.2
system (SAS Institute Inc., Cary, NC). The analysis was
carried out in three replications. Duncan’s multiple range
tests were applied to determine which of the differences
were significant (P \ 0.05).
Results and discussion
Proximate composition
Proximate analysis of the veggie burgers after 24 h of
refrigerated storage are presented in Table 1. Addition of
SC and MTGase had no significant effects on moisture, fat
content, and pH of burgers (P [ 0.05). Total protein and
ash of samples were significantly affected by the addition
of SC (P \ 0.05). Cofrades et al. (2011) evaluated the
quality features of low-salt restructured poultry containing
microbial transglutaminase and seaweed and found that the
addition of 3% Sea-Spaghetti and MTGase/caseinate had
no significant effects on moisture, protein, and fat contents
of the products. Pietrasik et al. (2007) investigated the
effect of addition of four non-muscle proteins (blood
plasma, sodium caseinate, soy protein isolate, and gelatin)
on pH values of the pork gels processed with microbial
transglutaminase. They showed that the pork gel containing
sodium caseinate had the highest pH values and MTGase
addition had no effect on pH of the samples. Canto et al.
(2014) also reported that MTGase had no effect on the pH
of the products.
Binding properties
One of the important features of burgers and other meat
products is the ability to hold water and other juices in the
product before and after cooking (Yang et al. 2007). In the
current investigation, the cooking loss and shrinkage were
used to measure the amount of juices that were lost during
cooking, and the expressible water was a measure of batter
stability of burgers before cooking and during storage.
Table 1 shows the impacts of SC and MTGase on the water
and fat binding properties of veggie burgers.
The percentage of expressible moisture is inversely
related to the water-holding capacity (WHC) that means,
the lower expressible moisture is usually associated with
the higher WHC. The amount of expressible water ranged
from 26.59 to 35.49%. The percent of expressible moisture
of the veggie burgers was lower than meat based products,
such as hamburger patties (Velioğlu et al. 2010) or crab
meat (Martı́nez et al. 2014). Previous researches
J Food Sci Technol (July 2017) 54(8):2203–2213 2207

7.00 ± 1.00AB
7.00 ± 1.00AB
5.00 ± 1.00BC

6.00 ± 1.00BC

6.00 ± 1.00BC
documented that the use of plant proteins plays a great role

8.00 ± 1.00A

8.00 ± 1.00A

8.00 ± 1.00A
5.00 ± 1.00C
4.00 ± 1.00C

5.00 ± 1.00C
5.00 ± 1.00C
to enhance water retention capacity and yield of meat
Diameter products (Pietrasik et al. 2007; Dzudie et al. 2002) and
probably for this reason WHC of the burgers was higher
than meat-based products. The addition of MTGase and SC
separately and in combination, significantly decreased the
AB

11.00 ± 1.00CD
AB

CD

3.00 ± 1.00AB
CD
BC

DE

EF

EF
14.00 ± 1.00A

8.00 ± 1.00E
Shrinkage (%)

expressible moisture of burgers. This parameter decreased

13.00 ± 1.00
12.00 ± 1.00

13.00 ± 1.00
11.00 ± 1.00
10.00 ± 1.00
9.00 ± 1.00

11.00 ± 1.00
9.00 ± 1.00
with increasing the levels of SC and MTGase (P B 0.05).
Thickness

Therefore the lowest expressible moisture was observed in

samples containing 2% SC and 0.75% MTG and the
highest EM was for the control sample. MTGase con-
tributes to the formation of covalent crosslinks between
Expressible moisture (%)

primary amines, such as lysine and glutamine residues in

SC and soy proteins, thereby enhancing the protein net-
work structure and expressible moisture of the various
35.49 ± 0.31A

H
C

34.68 ± 0.48B
31.61 ± 0.16E

26.59 ± 0.07I
34.11 ± 0.21
33.37 ± 0.33

34.87 ± 0.21
31.56 ± 0.17
30.01 ± 0.07
29.35 ± 0.08

29.16 ± 0.04
28.25 ± 0.06
protein-based products (Dzudie et al. 2002; Canto et al.
2014). However Pietrasik et al. (2007) showed that the
effect of MTGase on water retention was dependent on the
type of protein. Researchers reported that several proteins
such as, soy protein isolate or blood plasma in combination
6.70 ± 0.01A
A

6.69 ± 0.02A
A

6.72 ± 0.01A
A

6.72 ± 0.01A

with MTGase did not have any significant effect on water

6.70 ± 0.03
6.69 ± 0.03

6.71 ± 0.01
6.70 ± 0.02
6.71 ± 0.03
6.69 ± 0.02

6.72 ± 0.03
6.71 ± 0.02

retention ability of pork gel, but sodium caseinate and

gelatin were good substrates for MTGase and improved
pH

water retention of the gels. Pietrasik (2003) reported that

the addition of MTGase had no significant effect on water
holding of pork gel, although MTGase promoted the
17.16 ± 0.06A
A

17.15 ± 0.01A
A

17.12 ± 0.02A

17.15 ± 0.07A
A
17.13 ± 0.01
17.17 ± 0.01

17.17 ± 0.09
17.11 ± 0.01
17.19 ± 0.06
17.21 ± 0.06

17.16 ± 0.07
17.1 ± 0.14

hardness of gels at higher levels of sodium caseinate. These

contradictory results clearly demonstrate that the positive
Fat (%)

Values in columns with different superscript letters are significantly different (P \ 0.05)

effect of MTGase on water binding properties of protein

based products were dependent on the type of protein
substrate and the selected preparatory steps (Krintiras et al.
3.53 ± 0.02A
A

3.55 ± 0.02A
3.00 ± 0.00C
C

3.07 ± 0.04C
B

2015).
3.06 ± 0.06
3.04 ± 0.06

3.08 ± 0.01
3.08 ± 0.02
3.09 ± 0.03
3.11 ± 0.09

3.52 ± 0.03
3.56 ± 0.03

Shrinkage of cooked burgers is of particular importance,

Ash (%)

as it not only destroys the figure of a product, but can also

impact the economic aspect of meat and analogues meat
products (Li and Wick 2001). The percentage of shrinkage
Table 1 Proximate and physical properties of veggie burgers

varied between 8 and 14% (based on diameter). Overall,

14.04 ± 0.03A
A

14.07 ± 0.04A
12.45 ± 0.64C
C

12.62 ± 0.03C
B

B
12.62 ± 0.01
12.48 ± 0.04

13.25 ± 0.37
13.29 ± 0.23
13.33 ± 0.17
13.36 ± 0.28

14.05 ± 0.04
14.07 ± 0.06

the percentage of shrinkage in veggie burgers was smaller

Protein (%)

SC sodium caseinate, MTG microbial transglutaminase

than that reported by others in the meat-based products

(Ahhmed et al. 2009a, b; Velioğlu et al. 2010). Velioglu
et al. (2010) studied the effects of fat (15–30%), water
(10–20%) and textured soy protein (3–9%) content on the
50.12 ± 1.73A
A

50.87 ± 1.47A
A

50.42 ± 2.14A
A

51.50 ± 1.26A

shrinkage of hamburger patties after cooking. They

Moisture (%)

50.59 ± 2.46
50.81 ± 1.59

50.36 ± 1.84
51.22 ± 1.09
50.83 ± 1.63
51.40 ± 1.20

51.48 ± 0.94
51.48 ± 1.12

reported that both fat and water content had significant

effect on shrinkage and presence of textured soy protein in
formulation is the main factor for minimizing fat and
moisture loss. The results showed the application of
increased SC levels (from 0 to 2%) had no significant effect
SC0 ? MTG 0.75

SC1 ? MTG 0.25

SC0 ? MTG 0.25

SC1 ? MTG 0.75

SC2 ? MTG 0.25

SC2 ? MTG 0.75

SC0 ? MTG 0.5

SC1 ? MTG 0.5

SC2 ? MTG 0.5

on percentage of shrinkage of burgers (P [ 0.05) but 0.5

and 0.75% MTGase reduced the percentage of shrinkage of
Treatment

veggie burgers (P \ 0.05). No information has been found

Control

on the role of MTGase and SC addition on parameters of

SC1

SC2

processed veggie burgers. The results of the studies on the

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25.75 ± 2.02A
25.84 ± 2.34A
25.24 ± 1.47A
24.21 ± 2.64A
25.14 ± 3.89A
25.14 ± 5.05A
24.64 ± 4.11A
26.57 ± 2.40A
27.11 ± 5.27A
26.98 ± 6.33A
26.04 ± 5.24A
28.74 ± 4.44A
effect of MTGase and SC in restructured meat products are
contradictory. Su et al. (2000) reported that physical
entrapment of fat globules within the hydrated soy protein
or SC (2%) in the meat batters stabilized the meat emul-

b*
sions during cooking and decreased shrinkage and cooking
loss. Pietrasik (2003) found that the presence of increasing

16.191 ± 3.11A
16.43 ± 3.11A
A

15.37 ± 1.96A
16.94 ± 2.41A
A

A
levels of MTGase (0–0.6%) decreased the cooking loss in

18.11 ± 2.31

17.71 ± 2.69
16.29 ± 1.91
15.68 ± 1.80
14.14 ± 2.84
15.69 ± 2.68

18.37 ± 2.29
15.44 ± 1.81
cooked pork gels. However, Herranz et al. (2013) reported
that the binding properties were strongly influenced by
sodium content in processed muscle foods. Higher ionic

a*
strength enhances electrostatic repulsions and causes a
loosening of the myofibrillar structure. Cofrades et al.

40.56 ± 2.32AB

42.43 ± 3.95AB
DC
BC
45.57 ± 2.21A
A

D
39.43 ± 1.11B

C
44.55 ± 3.62

45.21 ± 2.63
42.92 ± 2.34
41.56 ± 2.16
41.10 ± 1.06
44.19 ± 3.24

39.86 ± 3.36
37.57 ± 2.90
(2011) also reported that when MTGase/caseinate was used
in formulations with the low salt content products it did not
affect percentage of shrinkage.

L*
Color measurement

A
C
G

922.56 ± 2.31D
H

945.25 ± 20.15
1032.25 ± 23.69
B
Cutting force (g)

886.50 ± 2.83E
822.89 ± 3.14F

F
659.05 ± 6.22I
789.82 ± 9.30

766.50 ± 1.41
922.56 ± 2.31
894.50 ± 2.12
991.25 ± 1.77
747.24 ± 3.01
Table 2 shows that the samples did not differ with respect to
redness (a*) and yellowness (b*) (P [ 0.05). However,
addition of 0.5 and 0.75% of MTGase reduced the lightness
(L*) of samples significantly. Addition of SC had no sig-
nificant effect on L* parameters of burgers but there was a
synergistic effect on decrease in lightness of burgers when
Chewiness (g.mm)

the enzyme and SC were applied at the same time (P \ 0.05).

HG

FG
H

10.61 ± 0.15D

A
C

B
9.50 ± 0.05E

E
8.21 ± 0.05F
5.10 ± 0.04I
7.08 ± 0.04

6.46 ± 0.14
9.31 ± 0.36
11.87 ± 0.48
13.53 ± 0.45
7.68 ± 0.13

13.03 ± 0.23
14.86 ± 0.92
The brown color of cooked burgers is attributed to the heat-
induced denaturation of proteins. MTGase contribute to
promoted denaturation of protein molecules (Canto et al.

Values in columns with different superscript letters are significantly different (P \ 0.05)
2014). Probably for this reason, for the same frying time,
veggie burgers which were treated with MTGase (0.5 and
Springiness (mm)

0.75%) were darker (lower L* value) and appeared more AB

CD

AB
3.32 ± 0.06DE
3.51 ± 0.04BC

DE

BC

3.81 ± 0.18BC
FE
2.23 ± 0.02G

A
cooked than those made without MTGase (control sample).
F
3.13 ± 0.18

2.99 ± 0.01
3.38 ± 0.25
3.71 ± 0.14
3.96 ± 0.03
3.58 ± 0.04

3.95 ± 0.01
4.19 ± 0.20
No reports were found regarding the effect of MTGase and
SC addition on veggie burgers color and the results of other
studies on various meat products were remarkably contra-
dictory. Several researches documented that MTGase levels
D

0.44 ± 0.01D

had no effect on the color of various cooked meat products

Cohesiveness

0.46 ± 0.01C

0.48 ± 0.01B
0.39 ± 0.01E
Table 2 Textural and color parameters of veggie burgers

0.43 ± 0.01

0.43 ± 0.01
0.46 ± 0.01
0.48 ± 0.01
0.50 ± 0.01
0.44 ± 0.01

0.50 ± 0.01
0.51 ± 0.01

(Canto et al. 2014; Cofrades et al. 2011). However, Min et al.

SC sodium caseinate, MTG microbial transglutaminase

(2008) observed that b* values increased with the addition of

MTGase in catfish patties. The differences between various
reports could be attributed to the differences in the formu-
lations (level of protein, fat, the addition of water, and other
447.00 ± 24.40H

2101.50 ± 57.98C
F

L* lightness, A* redness, b* yellowness

D

A
B
1339.25 ± 44.90

1737.00 ± 41.72
1454.50 ± 12.02
1562.83 ± 0.21E
1605.29 ± 7.12E
F

F
1471.50 ± 0.08

1471.50 ± 4.24
1709.00 ± 2.83

2227.50 ± 7.78
2777.00 ± 9.90

ingredients) and process of cooking of the various analogues

Hardness (g)

meat products.

Texture analysis

Raw veggie burgers

SC-1 ? MTG-2.5
SC0 ? MTG 7.5
SC0 ? MTG 2.5

SC2 ? MTG 2.5

SC2 ? MTG 7.5

Sc1 ? MTG 7.5
SC-1 ? MTG 5
SC0 ? MTG 5

SC2 ? MTG 5

The changes in texture features of raw burgers as a result of

Treatment

SC and MTGase addition are presented in Table 2. The

Control

results of texture profile analysis (TPA) indicated that the

SC1

SC2

incorporation of SC and MTGase had significant effects on

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hardness, cohesiveness, springiness, and chewiness of the
samples (P \ 0.05). Presence of SC (from 0 to 2%) did not
have any significant effect on cohesiveness of burgers. The
effect of MTGase addition on measured textural properties
of raw burgers was more than those produced without
MTGase (P \ 0.05). The addition of MTGase to formula-
tions enhanced the cohesiveness of batters and improved
their hardness, springiness, and chewiness. SC and MTGase
addition showed synergistic interaction effects on TPA of
samples, such that the veggie burgers made with MTGase
plus SC had higher hardness, chewiness, fracturability, and
cohesiveness values than other samples (P \ 0.05). The
highest textural properties were obtained in the presence of
both SC-2% and MTG-0.75% treatment. The textural
characteristics and WHC of protein based products can be
improved by incorporation of sodium caseinate and micro-
bial transglutaminase (Cofrades et al. 2011). In fact, for-
mation of large polymeric protein aggregates due to cross-
linking of adjacent protein molecules could be catalyzed by
MTGase, thereby improving functional and textural char-
acteristics, specially cohesiveness of the food products
(Martı́nez et al. 2014). The texture analysis of noodles [plant
protein based product, prepared by wheat flour, soy protein
isolate (SPI), and microbial transglutaminase] showed that,
MTGase incorporation has a positive effect on hardness and
chewiness of the noodles. The increases in hardness,
springiness, and chewiness of batters upon MTGase treat-
ment were related to the formation of strong and tight pro-
tein network that could trap the starch granules of the wheat
flour, limiting extra swelling of starch granules and
inhibiting the softening of the texture (Aalami and Leela-
vathi 2008). Moreover, the gel forming ability of soy protein
is intensified by MTGase and consequently is improved the
network density of the soy protein based product (Gan et al.
2009).
Cooked veggie burgers
As Table 2 indicates, cutting force (hardness) of cooked
burgers was lower than those of raw samples. The
decreases in textural parameters and cutting force can be
related to denaturation of proteins during cooking process
which led to a decrease in water holding capacity, an
increase in cooking loss and shrinkage of the protein net-
work (Velioğlu et al. 2010). Changes in concentration of
SC (from 0 to 2%) significantly increased hardness and
force for fracture of batter values (from 659 to 747.24 g).
MTGase was more significantly effective in improving
textural properties than SC and this effect was dependent on
the enzyme concentration. Combination of SC and enzyme
provided synergistic effect on hardness and fracture values
of samples. The observed increase in textural features is
attributed to a much stronger protein network formed with
2209
the addition of MTGase (Canto et al. 2014). Also, the heat
processing of samples allows protein unfolding and exposure
of reactive groups ultimately improves tenderness of the
product (Canto et al. 2014). Our results support the findings
of several previous investigations which indicated that the
addition of MTGase alone or in combination with SC could
enhance textural properties of various protein based products
(Gan et al. 2009; Pietrasik 2003). However, Cofrades et al.
(2011) reported that the addition of MTGase/caseinate did
not have any significant effect on improving texture of low-
salt restructured poultry. This could be due to the fact that the
effect of MTGase on protein network is species specific
(Ahhmed et al. 2009a, b). The poor texture and light color of
conventional veggie burgers (control sample) were critical
factors, which negatively affect consumer acceptance. Pro-
duction of veggie burgers by using MTGase/caseinate pro-
teins improves the texture and color properties of burgers
which had profound influence on the acceptance of the end
product.
Electrophoretic analysis
Electrophoresis experiments provide a denatured (SDS)
and reduced (DTT) conditions to break down non-co-
valent and disulfide bonds. Therefore, this analysis
reveals covalent bonds between proteins. The Elec-
trophoresis profiles of soy burger batters treated with
0.0–0.75% MTGase and 0.0–2% SC are shown in
Fig. 2a. The results indicated that distinct protein bands
were not formed on the SDS–PAGE and formed
smearing (poorly resolved protein) on the gel. Smearing
is likely due to the presence of protein–carbohydrate
derivatives, which will have a wide range of molecular
weights and consequently produce a smear of bands
(Davis et al. 2007) However, three zones of different
molecular weights can be distinguished in the elec-
trophoresis profile of MTGase treated samples; (1) high
molecular weight zone (H zone: MW [ 200 kDa), (2) a
smear of middle molecular weight protein (M zone) and
(3) low molecular weight zone (L zone: MW \ 15 kDa).
As seen in Fig. 2a, after enzymatic treatment, changes in
the lanes were evident. All concentrations of MTGase
treated samples showed H zone on the top of the gel,
which was formed by polymerization of protein com-
ponents in batter induced by MTGase, while H zone was
not found in the control samples. In the lanes corre-
sponding to the samples treated with MTGase, the
polymer fraction (H zone) increased, while the smear (M
zone) and monomer fractions (L zone) were less dense.
Protein migration in electrophoresis experiment can
mainly be supported by both hydrodynamic size and net
charge. Since the SDS gives a negative charge to pro-
tein, the higher electrophoretic mobility mainly relies on
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2210 J Food Sci Technol (July 2017) 54(8):2203–2213

Fig. 2 a Electrophoretic patterns of soy burger batters incubated with E microbial transglutaminase enzyme. b Line chart representing
MTGase and SC. MW molecular weight standards (in kDa), Control comparative effects of MTGase on different substrates
soy burger not-treated with MTGase, SC sodium caseinate,

the hydrodynamic size of protein molecule, which is and (2) low molecular weight fraction (b-conglycinin). The
changed by intramolecular cross-linking that catalyzed glycinins (11S) are hexamer, each composed of two trimers
by MTGase. In other words, large molecular components of three monomers subunits. Each monomer in turn is made
formed by MTGase with or without sodium-caseinate of an acidic and a basic polypeptide chains linked by
could not migrate well on gel and provided an extra disulfide bonds. Beta-conglycinins (7S) are trimers forrmed
band on the top of the gel (Djoullah et al. 2016; Kilic by various combinations of three polypeptide subunits: the
2003). When SC was added to the burger formulation, H a’, a, and b subunits. When soy protein was incubated with
zone was also not found (lanes 2 and 3). Addition of SC MTGase following electrophoretic changes were
brings about non-covalent interaction between SC and detectable:
other proteins of the burgers, thereby enhancing the
1. A progressive decrease of the subunits of 7 s globulin
mechanical properties of burgers, a process which is
and finally the disappearance of a9 subunit band at
probably disrupted by DSD. Similar result is reported by
0.75% enzyme. The attenuation of these bands might
Hu et al. 2015 on the effect of curdlan on SDS–PAGE
be due to the formation of intramolecular covalent
pattern and the texture properties of hairtail (Hu et al.
bonds in the polypeptide chains and production of a
2015). These authors suggested that hydrogen bonds
more compact protein structure (Djoullah et al. 2016).
between protein molecules were intensified with the
2. The attenuation of acidic and basic subunits (A and B,
increased level of curdling in hairtail gel and modified
respectively) of glycinin fraction in the enzyme treated
the gel texture.
sample and the elimination of A3 and B3 bands at
Also Fig. 2a shows that polymer content increased as a
0.75% MTGase level. Other studies have shown that at
function of the MTGase level. The greatest polymerization
the ionic strengths of 0.03–0.5, the acidic subunits
was appeared in the lanes corresponding to 2% SC and
were laid outside the glycinin complex and were more
0.75% MTGase. This result was shown to be concomitant
significantly affected by the enzyme, but the basic
with improved physical properties of the burgers (Table 2).
subunits were present in the interior of the glycinin
The increase of SC level in combination with MTGase
molecule and remained resistant to the MTGase (Wu
resulted in increasing polymer content, suggesting that the
et al. 2016). However, in the current research, elim-
SC is a good substrate for MTGase. Results of Fig. 2b
ination of B3 band was observed, probably as a result
supported this hypothesis that MTG reacts with soy, gluten,
of a longer incubation time (1 h at the room temper-
and SC. From this figure it can be concluded that MTGase
ature followed by 24 h at 4 °C) which made B3
had the largest effect on SC 2%.
subunits more accessible to enzyme. Tang et al. 2006
reported that after 240 min treatment of soy protein
Electrophoresis patterns of soy protein
with MTGase the density of basic subunits signifi-
cantly decreased but this decline did not lead to
The effect of different MTGase concentration on SDS–
removal of these bands. These authors considered that
PAGE patterns of soy protein was also investigated
the SDS–PAGE patterns of the protein were relying on
(Fig. 3a, b). Soy proteins mainly consist of two important
the incubation time (Tang et al. 2006).
fractions; (1) High molecular weight fraction (glycinin),

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J Food Sci Technol (July 2017) 54(8):2203–2213
Fig. 3 Electrophoretic patterns of soy and gluten proteins incubated
with MTGase and SC. a Electrophoretic patterns of soy proteins
incubated with different concentrations of transglutaminase. b Soy
protein bands at different concentrations of MTGase. B1–B8 protein
bands numbered from bottom to top of the gel, respectively, B9
protein polymers formed by MTGase. c Electrophoretic patterns of
3. The formation of the large polymers (at the top of the
gel; MW [ 200 kDa) in the samples treated with
MTGase. When SPI was incubated with MTGase, the
monomer fraction was decreased while the polymer
fraction was increased (Djoullah et al. 2016).
This shows that SPI is a substrate for transglutaminase
and can participate in the polymerization (Fig. 2b).
Electrophoresis pattern of gluten
Gluten separated into three fractions on the SDS–PAGE
based on their MW. These are the high-molecular weight
subunits of glutenin (HMW-GS), a medium-molecular-
weight group (omega-gliadins) and a low-molecular-weight
group including a and c-gliadins (LMW-GS) (Aminlari and
Majzoobi 2002). As seen in Fig. 3c, d electrophoretic pat-
tern was slightly changed after MTGase treatment. The
2211
gluten incubated with transglutaminase; d gluten bands at different
concentrations of MTGase. B1–B6 protein bands numbered from
bottom to top of the gel, respectively. B7 protein polymers formed by
MTGase. E microbial transglutaminase enzyme, S soy protein,
G gluten
medium-molecular-weight group (omega-gliadins) and
LMW, showed density attenuation in samples containing
MTGase as a function of enzyme level. The results of SDS–
PAGE analysis in the present study are in agreement with
the findings of several studies (Ahirwar et al. 2015). How-
ever, the production of high-MW bands in MTGase-treated
samples was notably non-extensive. Three explana-
tions for this phenomenon are suggested; (1) some peptides
(especially \ 14 kDa) in gluten remained in a compact
structure, and a part of glutamine residue was not accessible
for MTGase (2) Wheat protein is glutamine-rich and lysine-
poor consequently it was resistant to intramolcular cross-
linking induced by MTGase (Chin et al. 2009); (3) gluten
contained cysteines which contributes to the formation of
disulfide bonds (Aminlari and Majzoobi 2002).
In conclusion, SDS–PAGE patterns show that microbial
transglutaminase catalyzed intra- and intermolecular cross-
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2212 J Food Sci Technol (July 2017) 54(8):2203–2213

Table 3 Sensory properties of veggie burgers as influenced by added MTG and SC

Treatment Color Taste Appearance Mouth feel Overall acceptability

Control 2.80 ± 1.00FG 2.90 ± 0.71E 2.65 ± 0.48G 2.30 ± 0.80F 2.90 ± 0.71E
DEF CD DEF D
SC0 ? MTG 2.5 3.40 ± 0.50 3.60 ± 0.50 3.40 ± 0.50 3.40 ± .50 3.45 ± 0.51CD
ABCD AB CDE BCD
SC0 ? MTG 5 3.90 ± 0.44 4.15 ± 0.48 3.60 ± 0.59 3.70 ± .47 3.90 ± 0.44BC
SC0 ? MTG 7.5 4.15 ± 0.48AB 3.80 ± 0.41ABC 3.95 ± 0.39ABC 3.80 ± .41ABCD 4.05 ± 0.22AB
EFG DE FG EF
SC1 3.10 ± 0.85 3.20 ± 0.41 2.95 ± 0.51 2.60 ± .50 3.20 ± 0.41DE
BCDE BCD BCDE CD
SC-1 ? MTG-2.5 3.65 ± 0.48 3.65 ± 0.48 3.65 ± 0.48 3.60 ± .50 3.70 ± 0.47BC
A A A A
SC-1 ? MTG 5 4.40 ± 0.50 4.25 ± 0.44 4.35 ± 0.48 4.20 ± .41 4.50 ± 0.51A
AB ABC ABCD AB
Sc1 ? MTG 7.5 4.25 ± 0.44 3.85 ± 0.36 3.90 ± 0.30 4.15 ± .36 4.10 ± 0.30AB
SC2 2.50 ± 0.68G 3.20 ± 0.41DE 3.25 ± 0.44EF 2.90 ± .30E 2.90 ± 0.30E
CDE CDE BCDE ABC
SC2 ? MTG 2.5 3.50 ± 0.51 3.40 ± 0.50 3.65 ± 0.58 3.95 ± .51 3.50 ± 0.51CD
ABCD CD AB AB
SC2 ? MTG 5 4.00 ± 0.45 3.50 ± 0.51 4.15 ± .36 4.10 ± .30 3.90 ± 0.30BC
SC2 ? MTG 7.5 4.10 ± 0.44ABC 3.50 ± 0.51CD 3.85 ± .58ABCD 4.00 ± .32ABC 4.00 ± 0.32B
Values in columns with different superscript letters are significantly different (P \ 0.05)
SC sodium caseinate, MTG microbial transglutaminase

linking reaction of proteins from one source or two sources
used in this research.
Sensory properties
The sensory characteristics (color, taste, appearance, mouth
feel, and overall acceptability) are presented in Table 3. The
sensory analysis was performed based on 5-point hedonic
scale and scores higher than ‘3’ are considered acceptable.
The sensory scores of every parameter for treated samples
were higher than 3, as compared to the control. There was a
significant difference (P \ 0.05) in color, taste, appearance,
mouth feel, and overall acceptability between treated and
control samples. These results are in agreement with other
reports, implying the addition of MTGase and SC enhances
acceptance of various meat analogues samples. It seems that
MTGase, through formation of crosslinks between protein
molecules results in improvement of texture and quality
attributes of veggie burgers, such as WHC, expressible
moisture, cooking loss and shrinkage, thereby enhancing the
sensory properties of samples and improving overall accept-
ability of veggie burgers.
Conclusion
High consumption of red meat and its products poses
health problems. Substitution of red meat with plant pro-
teins might be beneficial to the consumer’s health. The
purpose of this research was to produce veggie burgers
based on soy protein and to study the effects of microbial
transglutaminase/caseinate (MTGase/SC) on physico-
chemical and sensory properties of veggie burger. The
results indicated that the addition of MTGase alone or in
combination with SC catalyzes the cross-linking reaction
between primary amines and forms high-molecular weight
polymers and subsequently, improves textural parameters
and color of veggie burger and enhances the acceptance of
the product.
Acknowledgements Funding was provided by Shiraz University.
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